Metal-organic framework materials (MOFs) have been widely used in food contamination adsorption and detection due to their large specific surface area, specific pore structure and flexible post-modification. MOFs with specific pore size can be targeted for selective adsorption of some contaminants and can be used as pretreatment and pre-concentration steps to purify samples and enrich target analytes for food contamination detection to improve the detection efficiency. In addition, MOFs, as a new functional material, play an important role in developing new rapid detection methods that are simple, portable, inexpensive and with high sensitivity and accuracy. The aim of this paper is to summarize the latest and insightful research results on MOFs for the adsorption and detection of food contaminants. By summarizing Zn-based, Cu-based and Zr-based MOFs with low cost, easily available raw materials and convenient synthesis conditions, we describe their principles and discuss their applications in chemical and biological contaminant adsorption and sensing detection in terms of stability, adsorption capacity and sensitivity. Finally, we present the limitations and challenges of MOFs in food detection, hoping to provide some ideas for future development. Salmonella, have been reported , food additives , prohibited additives , harmful substances produced during food processing and storage . Physical contamination mainly includes foreign substances and radioactive material contamination. These food risk factors in raw materials, processing, transportation, preparation, storage, and supply may produce harm at any time . In recereported . Thereforeported . Most ofreported . To overreported . Metal-oreported . In addireported . Thereforeported  for sampreported .MOFs are a class of hybrid porous materials with infinite topological structures. At present, more than 20,000 kinds of MOFs have been reported , and theThis paper reviewed the recent research progress in the adsorption and detection of food contamination from the perspective of different metal ion MOFs. Based on the preliminary literature research, the paper listed several common types of MOFs, including zinc-based, copper-based, and zirconium-based MOFs , which a2) by linking organic linking ligands to metal ions/clusters (secondary units SBUs). MOFs have a well-defined skeletal structure similar to that of zeolites. Unlike heterogeneous carbon materials, however, they have a very uniform pore distribution. In addition, the addition of functional groups  to organic linkers, the selection of different metals depending on the application and the flexible adjustment of the electronic properties of the pore surface are difficult to achieve with commercially established materials such as zeolites and activated carbons , was first introduced by  carbons . Rigid M carbons  so that  carbons . Flexibl carbons .4(BDC)3 (DMF)8C6H5Cl, DMF = dimethyl formamide], also named as IRMOF-1, was first synthesized by 2+ to form a three-dimensional material in octahedral form, the specific surface area of MOF-5 is as high as 2,900 m2/g. The commonly studied ZIF-8 comprises transition metal ions Zn2+ coordinated with imidazole groups or imidazole derivatives. In the reaction, the N atom on the rigid imidazole ring loses its proton and coordinates with the transition metal ion. The combination of metal ions with imidazolyl or imidazole derivative ligands can produce different structures of ZIFs under different conditions, achieving a diverse and tunable structure. Guest@ZIFs, which are a kind of composite materials with ZIFs as the main body to load functional guest molecules, are widely studied because of their ability to enhance some properties of the main material, such as catalytic, electrochemical and fluorescent activity (6 (μ3-O)4 (μ3-OH)4(CO2)12 metal ion cluster linked to phthalic acid. Metal ion clusters as nodes combining with organic ligands could greatly increase the porosity and specific surface area of the material, and UiO-66 also exhibits excellent stability. Due to their strong porosity and structural stability, Zr-MOFs have been intensively investigated in catalysis, molecular adsorption separation, drug delivery, fluorescence sensing, electrochemistry, and porous carriers  through the disruption of cell membrane integrity through the active sites on material surface to degrade proteins and fatty acids on bacterial membrane. Synergistic effects  can also2+ . Gwon etThe ability of MOFs as adsorbents can be attributed to their great structural potential, such as central metal ions, open metal sites, linkers, porosity, and functionalization/modification of MOFs. The main advantages of MOFs are their large specific surface area, huge porosity, and easy adjustment of pore size and shape from microporous to mesoporous scale, which can lead to high extraction efficiency . MOFs clHighly active sensors composed of nanocomposites typically have high adsorption capacity, large surface area, inter-crystalline mesoporosity and low electron transfer resistance . MOFs-baMetal ions are often used as signal tags in electrochemical assay systems because signals generated by chemical valence changes can be electrochemically detected, allowing some MOFs to exhibit excellent conductivity and high charge mobility. Many MOFs are involved in redox reactions. For efficient electrochemical sensors, two main points need to be satisfied: 1) high specificity of the signal tag and 2) high sensitivity and stability of the modified electrodes. MOFs and their composites can be designed as signal tags for electrochemical sensing because of their special structure, and water-stable MOFs are usually seen as long-term stable sensors . In addi2) and stability in organic solvents due to the strong bond between the central metal ion and the nitrogen atom in the ligand. ZIF-8, composed of zinc ions and 2-methylimidazole, has been extensively studied due to its unique properties such as large endopores and specific surface area   .2 framework and a large cage-like (∼12.5 Å) highly hydrophobic pore system connected by small windows (3/g), large surface area , and excellent stability in organic solvents, water and highly alkaline aqueous solutions  windows  (∼3.3 Å)olutions . ZIF-8 solutions . Yang etolutions . Therefoolutions . Abdi anolutions .3O4@AMP&ZnCl2@McAbs) for multi-target adsorb deoxynivalenol (DON) , zearalenone (ZEA), and T-2 and HT-2 toxins produced by Fusarium oxysporum. The results showed that the maximum adsorption amounts of Fe3O4@AMP&ZnCl2@McAbs per 100 mg composite were DON 688.26 ng, ZEN 864.98 ng, T-2 1,272.06 ng, HT-2 1,529.74 ng, respectively, and the recovery of the four mycotoxins decreased with the increase of the number of cycles. However, the decrease was insignificant, indicating the composites’ stability to some extent. The strong chemical and thermal stability of aflatoxin B1 (AFB1) make it more difficult to be removed from practical applications. Using reactive oxygen species with high reactivity is considered as an effective means to eliminate AFB1. Recently, Mycotoxins are secondary metabolites produced by fungal growth in food and feed, which are highly toxic and carcinogenic, resistant to high temperatures, chemically stable, and difficult to remove during general food processing. β-galactosidase  and glucose oxidase (GOx) for detected lactose to improve the quenching rate of fluorescence AuNCs. Since AuNCs are only 0.8 nm in size and have BSA on their surface, BSA-immobilized AuNCs can be co-encapsulated with β-Gal and GOx in ZIF-8 at room temperature. When lactose is in the system, lactose is first decomposed into galactose and glucose by β-Gal, and glucose is decomposed into gluconic acid and hydrogen peroxide (H2O2) by GOx in the presence of O2 in the air. The reaction between H2O2 and Fe2+ generates hydroxyl radicals, leading to the fluorescence quenching of AuNCs. Based on the above principle, the detection of lactose content in lactose-free milk had a detection limit of 41.67 μM at a signal-to-noise ratio of 3, which was much lower than the threshold value of lactose-free milk samples . As the long-term storage stability of enzymes is crucial for the utility of the sensor, the authors stored AuNCs/β-Gal/GOx at ZIF-8 and the free enzyme system  for 3 days, and found that the relative activity was 76.83% and 5.30% of the initial total activity, respectively, indicating that the ZIF-8 framework material established a tremendous digestive enzyme tolerance. Meanwhile, the quenching plastic rate of AuNCs/β-Gal/GOx@ZIF-8 fluorescence by lactose was increased to 3.4 times that of the free enzyme system.Luminescent metal-organic frameworks (LMOFs) have received increasing attention in sensing . Zn-baseE. coli O157:H7. In this system, cadmium sulfide quantum dots (CdS QDs) were encapsulated in ZIF-8 to construct a CdS@ZIF-8 complex with a core-shell structure. To specifically recognize O157:H7 cells, CdS@ZIF-8 particles introduced amino groups on the surface by wrapping them with polyethyleneimine (PEI) to enhance the interaction with antibodies. The biosensor had a linear range of 10–108 CFU/mL for O157:H7 detection and a detection limit of 3 CFU/mL (S/N = 3). The sensitivity of the proposed method was 16 times higher than that of the immunosensor using only CdS quantum dots as signal tags. The detection efficiency of the CdS@ZIF-8@PEI sensor was tested by spiking O157:H7 in milk. The results showed recoveries in the range of 94.3%–104.8%, indicating that the biosensor can be used to detect E. coli O157:H7 in actual samples.In a previous study , the autCopper is a variable valence metal in transition metals, which is relatively present in large quantities and cheaper than other transition metals. Copper ions have Lewis acidity and strong electrophilic capacity. Generally, Cu-based MOFs have a large specific surface area, diverse structure, and unsaturated coordination metal centers, which greatly enhance their application in electrochemistry . One of in situ synthesis of DMP-Cu at room temperature, and the modification of 2,5-dimercapto-1,3,4-thiadiazole (DMTZ) was found to increase the stability and effectiveness of the complex MOFs. The pH of the solution is one of the most critical factors affecting the adsorption performance. Mercury exists as Hg2+ when the pH is less than three and as Hg(OH)2 when the pH is more significant than 6.47. Both forms of mercury exist simultaneously in the pH range of 3–6.47  in the presence of H2O2. Furthermore, the presence of abundant amine groups on the surface of Cu-MOF nanoparticles allows for a simple modification of the S. aureus aptamer, therefore ensuring the selective recognition of S. aureus by Cu-MOF nanoparticles. By combining the Cu-MOF-catalyzed chromogenic reaction with aptamer recognition and magnetic separation, the detection limit for S. aureus detection was 20 CFU/mL. Recently, S. aureus detection using Cu-MOF, which reduced the detection limit of S. aureus to 5.2 CFU/mL 4 and a 1,4-benzenedicarboxylic acid ligand. Its thermal, mechanical and hydrolytic stability all up to 450°C is mainly attributed to the stable coordination bond between Zr4+ and the carboxylate ligand.Zr-based MOFs generally have better mechanical stability because they have high connectivity of Zr group clusters and strong Zr-O coordination bonds in the framework . Zr-base6 clusters and Coulombic gravitational force between these Zr cationic clusters and the anionic ligands (2 (2 (2) in milk . Adsorption kinetic study revealed that the adsorption kinetic model of MOFs was more consistent with pseudo-second-order adsorption kinetics, indicating that the rate-limiting step of the adsorption process was chemisorption . Wang e in milk . The satsorption . There wwith SM2 . The slo2) using chitosan (CS) modified UiO-66-NH2, which solved the problem of agglomeration of UiO-66-NH2 in practical applications and increased its contact area with heavy metal ions, achieving a stronger adsorption capacity. The UiO-66-NH2 attached to the main chain of CS by covalent bonding did not reduce the active adsorption sites but noticeably improved the dispersibility and preserved the morphology of the MOFs as much as possible. The adsorption performance of Pb(II) showed that the maximum adsorption capacity reached 555.56 mg/g. After five cycles, the adsorption capacity still reached 74.8% of the first adsorption capacity, indicating that the composite MOFs material had an excellent reusability. In another study, 2 with polyethyleneimine (PEI), and the prepared composite PEI@UiO-66-NH2 exhibited even better adsorption performance for Pb(II)  .6 clusters in BUT-16 to form a large number of hydrogen bonds. However, BUT-17, which has the same spatial configuration, has a tiny difference between the ligands used to build MOFs . As a result, the O atom in the adsorbed DON molecule can only form a hydrogen bond with the H atom on the ligand in BUT-17, therefore showing a tremendous difference in the adsorption. Meanwhile, such a slight difference in the ligands made the angle of the planar rings in the ligands different and allowed the DON molecules to enter the Zr6 cluster in BUT-16 more easily. By testing in water samples, BUT-16 demonstrated a high adsorption capacity of 46 mg/g for DON molecules.Most conventional MOFs are difficult to be biologically active as a chemical material. Recently, Based on molecular imprinting technique, 6 ion cluster in water plays a crucial role in mycotoxin detection. in situ hydrothermal synthesis. Such a bifunctional hybrid sponge not only retained the great mechanical processing properties of MF sponge, but also had a high tolerance to large deformation. At the same time, the pore structure of the preserved Zr-LMOFs crystals allowed the guest molecules to enter well inside the cavity, enabling the detection of AFB1 with a relatively high sensitivity (LOD = 1.6 ppb) and a wide linear range of 0.1–50 μM. It was further found that the analyte-sensor interaction played a crucial role in the efficient quenching. Moreover, AFB1 was rapidly enriched in the cavity of Zr-LMOF, producing stronger analyte-sensor interaction and higher-order π-π interaction, which could accelerate the electron transport and generate a greater quenching effect for sensitive detection. Therefore, based on this property, it is feasible to develop hybrid materials based on Zr-LMOF for rapid detection and removal of toxic and hazardous substances.6 clusters of Zr-based MOFs give them high chemical, thermal and mechanical stability compared to other materials. As can be seen from The Zr2-MIL-53 is a nanoscale particle, it has both fluorescence and second-order scattering signals. The intrinsic fluorescence signal of CIP overlaps with NH2-MIL-53, which enhances the background signal and is not conducive to detection. Therefore, the authors modified it with 8-hydroxy-2-quinolinecarboxaldehyde (HQCA), and the modified HQCA-MIL-53 showed almost no fluorescence properties compared to NH2-MIL-53, but still exhibited scattering properties. 2O4@SiO2@MIL-101(Cr)-NH2 for magnetic solid phase adsorption (MSPE) of pesticide residue acetamiprid from fruit juice. The adsorbent utilized the synergistic effects of the high specific surface area of MIL-101(Cr), magnetic properties of CoFe2O4, and specific molecular recognition of the aptamer, and showed a strong performance in the extraction of aminopyralid with a maximum adsorption capacity of 3.5 mg/g. In recent years, cyclodextrin metal-organic frameworks (CD-MOFs) based on cyclodextrin (CD) have been widely investigated not only for their typical edible, regenerative, and biodegradable properties, but also for their large surface area, controllable pore size, and high adsorption properties on gases. 3-PEDOT/CD-MOF/GCE) using γ-CD-MOF, MoO3 and a flexible conducting polymer poly  (PEDOT). The in situ growth of PEDOT on MoO3 nanorods effectively improves the stability, dispersion, and electrical conduction efficiency of PEDOT. Meanwhile, the cavities in CD-MOFs can absorb a large amount of LU and increase the local concentration of LU on the electrode surface, which further amplifies the current signal. Under the optimal conditions, the sensor exhibits a wide linear range of 0.4 nM–1800 nM and a low detection limit of 0.1 nM (S/N = 3).In addition to the several metal-based MOFs mentioned above, many other MOFs materials have been investigated for the adsorption and detection of food contamination. Among them, Co-based MOFs are fast developing and have been gradually applied to food detection. 2+/Cu, Cu-based MOFs are electrochemically active, and the large number of electroactive sites in Cu-MOFs facilitates signal amplification, so they are used more in electrochemical sensors. Meanwhile, Cu ions have been studied more in food preservation due to their broad-spectrum antibacterial activity, such as loading mesoporous HKUST-1 on chitosan quaternary ammonium salt films to achieve good ethylene adsorption and antibacterial capacity  and have bulk rejection of large molecules, so precise control of pore size and volume for specific applications (including adsorption performance and sensing selectivity) is still to be development. Secondly, the functional versatility, structural flexibility and large specific surface area of MOF materials are their greatest advantages as adsorbent materials, compared to commercially available materials such as zeolites and activated carbon. However, some MOF materials still lack stability in aqueous solutions, which is an urgent issue in the development and utilization of MOFs. The stability of MOFs can be improved by compounding with different stabilizing materials. Furthermore, an important limitation of MOFs is the use of non-renewable raw materials for their synthesis, but this problem has recently been solved by the manufacture and use of γ-cyclodextrin-based MOFs. Currently, γ-cyclodextrin-MOFs are being developed as a new green and edible MOF and will be considered for future research in food packaging and food additives. Finally, some of the already commercialized MOFs  can be developed for specific applications depending on the metal ion. Zn-based and Zr-based MOFs materials have been widely employed in the detection of chemical contamination because of their stable skeleton structure as well as in fluorescence detection and analysis. Due to the redox reaction of Cucapacity . It can


Second, PIP and MTX were individually nanoformulated into F-127 pluronic nanomicelles (PIP-NMs) and F-127/P-105 mixed pluronic nanomicelles (MTX-MNMs), respectively, validated by several characterization techniques, and the re-investigated cytotoxicity of PIP-NMs and MTX-MNMs was fortified. Besides, the PIP-NMs/MTX-MNMs demonstrated further cytotoxicity enhancement. The PIP-NMs/MTX-MNMs combination was analyzed by flow cytometry to understand the cell death mechanism. Moreover, the in vivo assessment of PIP-NMs/MTX-MNMs was adopted through the Ehrlich ascites model, which revealed a significant reduction of the tumor weight. However, some results of the tumor markers showed that the addition of PIP-NMs to MTX-MNMs did not significantly enhance the antitumor effect.Breast cancer is a prevalent tumor and causes deadly metastatic complications. Myriad cancer types, including breast cancer, are effectively treated by methotrexate (MTX). However, MTX hydrophobicity, adverse effects and the development of resistance have inspired a search for new effective strategies to overcome these challenges. These may include the addition of a bioenhancer and/or encapsulation into appropriate nano-based carriers. In the present study, the anticancer effect of MTX was fortified through dual approaches. First, the concomitant use of piperine (PIP) as a bioenhancer with MTX, which was investigated in the MCF-7 cell line. The results depicted significantly lower IC Breast cancer is the most observable cancer type affecting females in both developing and developed countries ,2. GlobaMethotrexate (MTX) is an anticancer hydrophobic drug that exerts its cytotoxicity through the inhibition of dihydrofolate reductase (DHFR) enzyme that coverts the thymidylate synthase product; dihydrofolate to tetrahydrofolate. Consequently, this inhibition depletes the cells from thymidylate and prohibits the DNA and purine synthesis ,6. MTX hPiper nigrum) [Piperine (PIP) is a natural drug of piquant taste that is extracted mainly from black pepper ( nigrum) . Among i nigrum) , anti-in nigrum) , antihyp nigrum)  and anti nigrum) . Intrigu nigrum) ,31. Ther nigrum) , curcumi nigrum)  and resv nigrum)  as a sim nigrum) . To over nigrum) ,37.® are triblock copolymers consisting of variable chain lengths of polyethylene oxide (PEO) and polypropylene oxide (PPO). Due to their safety, biocompatibility, biodegradability and amphiphilic nature, they are used extensively to develop self-assembled nanoparticulate carriers as DDSs that enable the encapsulation of both hydrophilic and hydrophobic drugs [® are able to escape uptake by macrophage cells because of their PEO hydrophilic shell. Besides, their tiny nano-size allows their accumulation within the cancer tissue through the enhanced permeability and retention (EPR) effect. Moreover, pluronics® nanocarriers overcome drug resistance by the P-gp efflux system [Pluronicsic drugs ,39,40,41x system ,43,44.Herein, the anticancer effect of MTX against breast cancer was fortified through two different strategies . First, ® F127, pluronic® P105, MTX, triethylamine, methanol, acetone, dimethyl sulfoxide (DMSO), MTT and trypan blue were purchased from Sigma . Fetal bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25% trypsin-EDTA were obtained from Lonza (Belgium). PIP (molecular weight of 285.34 Da and purity 98%) was provided by Alpha Aesar .Pluronic® F127 and P105 (1:8 w/w) were dissolved in 5 mL methanol and 50 µL triethylamine. Then, a thin-film was formed with the aid of a rotary evaporator, followed with hydration by double volume of distilled water. The MTX-MNMs were prepared using the same procedure as the plain MNMs after adding a pre-determined amount (7 mg) of drug to the pluronics solution.Plain (drug-free) mixed pluronic nanomicelles (MNMs) and MTX-loaded MNMs (MTX-MNMs) were prepared by thin-film hydration method . Briefly® F127 only (40 mg) or mixed with a pre-determined amount (5 mg) of PIP were dissolved in 15 mL ethanol to develop plain NMs and drug-loaded NMs (PIP-NMs), respectively. Then, the ethanolic solution was added dropwise to a double volume of distilled water, followed by stirring overnight to evaporate the organic solvent [Both plain NMs and PIP-loaded NMs (PIP-NMs) were prepared by nanoprecipitation method . Either  solvent .−1.The particle size (PS), polydispersity index (PDI) and surface zeta potential (ZP) of plain MNMs, MTX-MNMs, plain pluronic F127 (NMs) and PIP-NMs were obtained by dynamic light scattering using a Zetasizer Nano ZS  at room temperature. The morphology of all the developed NMs was visualized by HR-TEM . Lyophilized samples were investigated by Fourier transform infrared (FTIR) spectroscopy to obtain the main peaks for the chemical characterization using universal ATR module  by VERTEX 70 FTIR spectrometer 16  in the range of 600–4000 cmi is the initial amount of drug, Drugf is the amount of free (unentrapped) drug.The entrapment efficiency (EE%) and loading capacity (LC%) of the drugs were determined by the UV-Vis spectrophotometry  at 310 nm and 342 for MTX and PIP, respectively. The following equations were applied:The release profiles of free MTX drug and MTX from the developed MTX-MNMs were determined using the dialysis bag method  in PBS of different pH values; 7.4 and 5.5. Briefly, the samples were dispersed in pre-determined volume of PBS (as donor chamber) and left in a shaker incubator (110 rpm) at the physiological temperature 37 ± 1 °C. At certain time intervals, a fixed volume of aliquots was withdrawn and replaced with fresh buffer. The amount of the released drug was quantified using UV-Vis spectrophotometer at the corresponding wavelength (310 nm).2 atmosphere for subculturing 2 to 3 times weekly. In a Corning® 96-well tissue culture plate, the cells (5 × 104 cell/well) were suspended and incubated for 1 day. Then, 12 serial concentrations of the tested formulation were added to the wells as triplicates. Six controls were used and were either the plain medium or 0.5% DMSO. After incubation for 1 day, the count of the viable cells was detected by MTT assay. Briefly, the media was replaced by 100 µL of RPMI-1640 without phenol red and the 10 µL of 12 mM solution were added to each well and incubated at 37 °C and 5% CO2 atmosphere for 4 h. Then, 85 µL of the media were discarded and replaced by 50 µL DMSO and incubated at 37 °C for 10 min. The number of the viable cells was determined by measuring the optical density at 590 nm using the equation:The in vitro MTT cytotoxicity assay of free drugs (PIP and MTX), PIP-NMs and MTX-MNMs, in addition to the mixtures consisting of free PIP/free MTX and the PIP-NMs/MTX-MNMs, was performed using MCF-7 cells (human breast cancer cell line). The cells were obtained from the American Type Culture Collection . The cells were cultured on RPMI-1640 medium treated with 10% inactivated fetal calf serum and 50 µg/mL gentamycin and kept in 5% COODt: the optical density of the cells treated with the formulationsODc: the optical density of the untreated cells (control)50 was calculated from plotting the dose response versus the formulation concentration using the software Graphpad Prism .The ICTM, Heidelberg, Germany) using the IC50/24 h. Briefly, the MCF-7 cells were cultured and incubated with the formulation for 1 day while the negative control cells were incubated with the plain medium. Afterwards, the cells were collected and washed with PBS twice. The cells were then washed by the binding buffer (100 µL) and FTIC-annexin V (1 µL) and incubated at 4 °C for 40 min. The cells were rinsed and resuspended in the binding buffer (150 µL) and DAPI (1 µL/mL in PBS) . Finally, the cells were tested by a flow cytometer .In order to understand the mechanism of the cell death by the investigated mixture (PIP-NMs/MTX-MNMs), annexin V-FITC assay was carried out using FITC-annexin V  was used to investigate the mixture-treated and the control (untreated) cells followed by staining by propodium iodide then analyzed by the cytometer. Then, CellQuest software  was used to calculate the cell cycle distribution.The effect of the mixture (PIP-NMs/MTX-MNMs) on cell cycle distribution was determined using MCF-7 cells by the flow cytometry. CycleTEST7 cells/mL) obtained from a female mice ascitic fluid. The mice were left for 2 weeks until a sensible solid tumor was obtained, with a size of more than 100 mm3. The mice were injected intraperitoneally on a daily basis for 3 weeks and divided into 3 groups:From the animal house of VACSERA, Egypt, 30 Balb/c mice were used to develop this test. The mice weight was in the range of 20 to 40 g each. The in vivo experiment was performed after receiving the approval from the research ethics committee for experimental and clinical studies at the Faculty of Pharmacy, Ain Shams University, Cairo, Egypt that complies with the United Kingdom Animals Scientific Procedures Act, 1986, and the European Union directive 2010/63/EU for animal experiments, as well as the ARRIVE guidelines. The Ehrlich tumor was induced in the mice by intramuscular injection in the right thigh of viable tumor suspension (1.25 × 10Group 1: negative control (received distilled water).Group 2: mice received MTX-MNMs  .Group 3: mice received PIP-NMs/MTX-MNMs  ,47.The tumor volume was monitored during the experiment at different periods  by vernier caliper  and calculated according to the equation:A: minor tumor axis, B: major tumor axisHowever, the tumor weight was detected after the animals’ sacrification.®, Houston, TX, USA) and Total SimpleStep ELISA® Kit, respectively as per the manufacturer’s instructions. DNA fragments were quantified as representative for the apoptotic cells using Apo-BrdU In Situ DNA Fragmentation Assay Kit  as per the manufacturer’s instructions. The AKT level was investigated using BCA test. Briefly, the tumor tissue was fragmented in lysis buffer by a homogenizer followed by centrifugation to isolate the supernatant. To allow cells lysis, sodium dodecyl sulfate (SDS) was added and the solution was then transferred to SDS-PAGE electrophoresis loaded with molecular weight marker. Then, the isolated proteins were transferred to a nitrocellulose membrane loaded with AKT antibodies  and left for 1 h. Then, the membrane was rinsed and incubated with horseradish peroxidase-conjugated secondary antibodies for 45 min. Finally, the samples were quantified by western blot through enhanced chemiluminescence (ECL). In an attempt to estimate the oxidative stress, MDA and SOD were detected by the Lipid Peroxidation Colorimetric/Fluorometric Assay Kit  and Biodiagnostic, Egypt, respectively, followed by colorimetric and absorbance measurement at 532 and 405 nm, respectively.After the animals’ euthanasia, the tumor tissue was obtained and cleaned with ice. Then, by polytron homogenizer, the tumor was homogenized and diluted by 0.05 M PBS (pH 7.4) to represent 10% of the solution. Afterwards, it was preserved as aliquots in −80 °C for further analyses to detect cyclin D1, p-38 MAPK (mitogen-activated protein kinase), apoptosis, AKT, malondialdehyde (MDA) and superoxide dismutase (SOD). Cyclin D1 and p-38 MAPK were detected using ELISA kit  for 24 h. Later, the tissues were dehydrated, set in paraffin blocks, and sliced at a thickness of 5 μm, transversely. Subsequently, the slides were stained with hematoxylin and eosin (H&E) and observed using Olympus BX43 light microscope linked with a digital camera (DP27) and CellSens dimensions software. The histological damage score was assessed at 200×, as previously described .For immunohistochemistry analysis, tissue samples were mounted on positive glass slides, deparaffinized, rehydrated and incubated with the primary mouse monoclonal antibody of TGF-β and Bcl-2 (1:100) according to the manufacturer’s instructions (Santa Cruz). ImageJ software was used to estimate the semi-quantitative of the area % of positive results (6 samples/group/200×).The preparation of the plain pluronic nanomicelles (NMs) and PIP-NMs was attempted by the nanoprecipitation method . On the The morphology of the developed plain and drugs-loaded nanomicelles was investigated by TEM . As can The EE% and LC% of each of the two drugs; PIP and MTX loaded into the developed PIP-NMs and MTX-MNMs, were estimated by UV-Vis measurement. In the case of the PIP-loaded NMs, the EE% and LC% were found to be 82% and 6%, respectively. While in the case of the MTX-MNMs, the EE% and LC% were determined to be 85% and 2.5%, respectively. These results are in consistency with the literature ,46.−1, respectively, -CO-N stretching at 1634 cm−1, symmetric and asymmetric stretching of C=C diene at 1612–1584 cm−1, respectively, aromatic stretching of C=C at 1518 cm−1, CH2 bending at 1428 cm−1, asymmetric and symmetric stretching of =C-O-C at 1254–1233 and 1045 cm−1, respectively, C-O stretching at 958 cm−1 and C-H bending at 847–802 cm−1. The plain pluronic NMs and MNMs spectra showed C-H stretching vibration peak at 2890 cm−1, O-H bending at 1342 cm−1 and C-O-C stretching vibration at 1110 cm−1 [−1 corresponding to the stretching of O-H of the carboxyl group, 2957 cm−1 of stretching of primary amine group (N-H), 1603–1647 cm−1 from C=O stretching from both the amide and carboxyl groups, 1494–1519 cm−1 from bending of N-H group, 1425–1229 cm−1 stretching of C-O of the carboxyl group, 885 cm−1 from O-H bending and 828 cm−1 from hydrogens on the aromatic ring [Free drugs (PIP and MTX), plain nanomicelles (NMs and MNMs) and the drugs-loaded NMs (PIP-NMs and MTX-MNMs) were scanned by FTIR to confirm the nanomicelles formation in addition to the physical drugs’ encapsulation, as shown in 110 cm−1 . From thThe release profiles of the free MTX and MTX-loaded MNMs (MTX-MNMs) at two different pH values (7.4 and 5.4) representing the physiological and cancer tissue pHs, respectively, are shown in 50 values of the free PIP at 24 h and of free MTX at 24 and 48 h were determined using serial concentrations ranging from 0 to 500 µg/mL. The free PIP IC50 at 24 h was found to be 183 μg/mL while the free MTX IC50 values at 24 and 48 h were found to be 213 μg/mL and 115.3 μg/mL, respectively . From the results, the IC50 values of the free MTX decreased significantly at both investigated time intervals (24 and 48 h) to be 45.23 μg/mL (p < 0.0001) and 25.84 μg/mL (p < 0.0001) at 24 and 48 h, respectively when compared to the MTX alone  on the cytotoxicity of MTX, the ICTX alone .50 values of the PIP-NMs at 24 h and MTX-MNMs at 24 and 48 h were determined  when compared to the free drugs counterparts. The IC50 of the PIP-NMs was decreased from 183 μg/mL to be 11.84 μg/mL (p < 0.0001). Additionally, the IC50 of the MTX-MNMs significantly decreased when compared to the free form of the drug at both time intervals; 24 and 48 h to be 3.47 μg/mL (p < 0.0001) and 2.099 μg/mL (p < 0.0001), respectively , as shown in 50 at 24 h was decreased about 5-fold to be 0.627 µg/mL (p < 0.0006). Additionally, the IC50 at 48 h revealed a much potent cytotoxic effect by decreasing the IC50 about 16-fold to be 0.128 µg/mL (p < 0.0001)  on the cytotoxicity of MTX-MNMs, the IC 0.0001) c.p < 0.0001), from 0.43% to 11.51%, and the late apoptosis increased significantly (p < 0.0001), from 0.17% to 3.68%. Besides, the necrotic cells were almost doubled from 1.01% to 2.27% (p < 0.0001). Based on the obtained results, the investigated PIP-NMs/MTX-MNMs combination was found to induce cell death through mainly enhancing both the early and late apoptotic stages  increased the cells arrest by about 1.5 times (from 13.19 to 18.67%). Thus, the cell cycle arrest occurs mainly in the G2/M phase, followed by the G0/G1 phase.Cell cycle analysis was performed to investigate the cell cycle kinetics induced by PIP-NMs/MTX-MNMs using the IC control . At the p < 0.05). On the other hand, there was no observable significant difference in the tumor volume between the groups that received MTX-MMNs and PIP-NMs/MTX-MNMs. Tumor weight was also determined at the end of the treatment duration, as per p < 0.05), with the PIP-NMs/MTX-MNMs-treated group exerting the most tumor weight reduction (p < 0.005) when compared to the MTX/MNMs-treated group.The mixture of the nanoformulated drugs was tested to evaluate any additional effect of the concomitant use of the bioenhancer (PIP)-loaded NMs (PIP-NMs) along with the MTX-MNMs as per the same ratio of the in vitro assessment. The tumor volume was determined at fixed days in all the mice groups, including the control group a. The tup < 0.005), Upon investigation of the cyclin D1 concentration, a significant reduction in its level was noted in both nanoformulations (MTX-MNMs and PIP-NMs/MTX-MNMs) when compared to the control group (p < 0.005), p < 0.05).The concentration of p-38 MAPK was determined in both the investigated nanoformulations-treated groups as compared to the control one. From the results, it can be elicited that there is a significant decrease of the p-38 MAPK level in both nanoformulations-treated groups (p < 0.05). Additionally, the addition of the PIP-NMs has enhanced the SOD concentration compared to the MTX-MNMs-treated group (p < 0.05). Consequently, the SOD elevated level upon treatment with the nanoformulation (MTX-MNMs) and the further elevation attained in the PIP-NMs/MTX-MNMs-treated group indicates the high number of the existing free radicals. These results are in concordance with the previously reported relation between MTX administration and the increase of SOD level [As an attempt to assess the oxidative stress, SOD and MDA concentrations were detected . The resOD level .p < 0.001). Besides, addition of the nanoformulated bioenhancer (PIP-NMs) to the MTX-MNMs further reduced the MDA level, though this further reduction was not statistically significant (p ≥ 0.05).Upon the assessment of the MDA level depicted in p < 0.005). However, the AKT level was almost the same in both the MTX-MNMs and PIP-NMs/MTX-MNMs-treated groups. As an additional attempt to investigate the apoptosis, the DNA fragmentation was assessed as apoptosis biomarker in the mammalian cells and considered as an indication of the anticancer treatment efficiency. From the results depicted in p < 0.05) and the control group (p < 0.005).The induced apoptosis was determined through phosphorylated AKT (pAKT) and tunnel assay determination by western blot . The obtp ≤ 0.01).The histological examination and observation of all groups are shown in Concerning immunohistochemical analysis, a strong positive expression of TGF-β and Bcl-2 that appeared as brown coloration of Ehrlich cells was observed in control group. Treatment with MTX-MNMs did not significantly reduce the expression of TGF-β  and Bcl-The aim of the study is to enhance the anticancer effect of MTX against breast cancer and evalute that in vitro and in vivo using the MCF-7 cell line and the Ehrlich ascites model, respectively. The MCF-7 cell line was used in this study, as it is the most common human breast cancer representative and the most studied in research . This en@ F127 NMs using a nanoprecipitation method with an EE% of 82%. In the case of MTX, it was loaded successfully (with EE% 85%) into mixed NMs (MNMs) of both pluronic@ F127 and P105 using a thin-film hydration method. MTX incorporation into MNMs was to avoid the large particle size obtained upon its encapsulation into pluronic@ F127 alone [@ F127 and P105 are amphiphilic polymers and their hydrophobic parts are very close in length; F127 PPO: 64 and P015 PPO: 56. Therefore, upon the preparation of the MNMs from these two pluronic polymers, they exhibited cooperative aggregation [Due to the hydrophobic nature of both drugs, PIP and MTX, their therapeutic bioavailability is limited. Therefore, they were encapsulated individually inside pluronic NMs. PIP was successfully encapsulated into pluronic27 alone . Pluroniregation , which fregation ,39,40,46regation .+ and the PEO of the pluronics. This interaction can lead to a decreased chain packing, and thus, loosen micelles structure due to charge repulsion [The release study was performed to identify the release rate of free MTX and the MTX from MTX-MNMs at two different pH values (7.4 and 5.4), representing the physiological pH and the cancer tissue pH, respectively, while maintaining the sink conditions. Free MTX exhibited a fast release rate, as almost all the drug was released after 4 h. However, the MTX-MNMs showed an initial burst release followed by a slower release pattern, with an observable faster rate at the acidic pH. In both pH values, the release was relatively fast within 24 h) due to the physical entrapment of the drug, which facilitates its liberation from the micelles [ h due toepulsion .50) so that the PIP amount did not affect more than 25% of the cells’ population [50 values of PIP and MTX were first detected individually. Afterwards, the IC50 value of the PIP/MTX mixture was reinvestigated after the addition of the half PIP IC50 to MTX, as previously mentioned. From the results, the addition of PIP enhanced the bioavailability and fortified the anticancer effect of free MTX. This was evidenced by the decrease of the IC50 value of MTX at 24 and 48 h after PIP addition as compared to the MTX alone. Consequently, the mixing of PIP with MTX as free drugs enhanced the cytotoxicity against MCF-7 cells.The bioenhancing effect of PIP on MTX anticancer activity was investigated by using both drugs in their free forms. PIP was added to MTX as per certain ratio . This nanoencapsulation was found to enhance the cytotoxic effect of both drugs due to the increased solubilization and thus, the better bioavailability. Besides, these individual nanoformulated drugs demonstrated lower IC50 values as compared to their free drugs counterparts, which may be attributed to the role played by the pluronic nanocarrier in enhancing the anticancer activity, where pluronics are able to escape the uptake by macrophage cells because of their PEO hydrophilic shell. Besides, the tiny nano-size of the pluronic nanocarriers allows their accumulation within the cancer tissue through the enhanced permeability and retention (EPR) effect. Moreover, pluronics nanocarriers have been reported to overcome drug resistance by P-gp efflux system [The ICx system ,43,44.50) so that the PIP amount did not affect more than 25% of the cells’ population. At the two investigated time intervals, the cytotoxicity was enhanced significantly as the IC50 value of the PIP-NMs/MTX-MNMs mixture decreased from 3.47 to 0.627 µg/mL and from 2.099 to 0.128 µg/mL, at 24 and 48 h, respectively, as compared to MTX-MNMs.The bioenhancing effect of nanoformulated PIP (PIP-NMs) on the anticancer activity of the nanoformulated MTX (MTX-MNMs) was investigated. PIP-NMs were added to MTX-MNMs as per a certain ratio , followed by mixing the PIP-NMs and MTX-MNMs together as per the ratio of their free drugs counterparts, and the cytotoxicity was then investigated. The nanomicelles formation was confirmed by various physical characterization techniques. From the cytotoxicity results, the pluronic encapsulation significantly enhanced the bioavailability of both MTX and PIP. Moreover, the mixing of the pluronic-encapsulated drugs significantly fortified the efficiency of the MTX as compared to either the free drug or the pluronic-encapsulated drug. The in vivo assessment was performed by adopting the Ehrlich ascites model, which confirmed the efficiency of MTX-MNMs treatment for 3 weeks, but did not confirm a significant enhancement by PIP-NMs addition. The future investigations would be directed to reveal a deeper molecular and mechanistic pathway investigations towards the optimum usage of the drug and the bioenhancer to achieve greater treatment efficiency.This study aimed to fortify the MTX efficiency against breast cancer through two approaches. The first one was through mixing MTX with the bioenhancer PIP, as per a certain ratio, and the anticancer effect was confirmed upon in vitro testing on MCF-7 cells as the IC


The premature and initial burst release of 5FU was prevented using 5FU-Alg-Np-HG. The release mechanism of 5FU from the 5FU-Alg-Np-HG diffusion was followed by swelling and erosion, as suggested by Korsmeyer-Peppas model. The prepared hydrogel proved to be non-irritant. Ex vivo permeation study across rat’s skin suggests that permeability of nanoparticles (5FU-Alg-Np) was higher than the 5FU-Alg-Np-HG . However, skin-related drug retention of 5FU-Alg-Np-HG was significantly higher than the 5FU solution, 5FU-Alg-Np, and 5FU-HG . This was due to swelling of hydrogels in the lower layers of skin where the temperature is 37 °C. The higher concentration of 5FU in the skin is helpful for treatment of local skin cancer, such as melanoma, and actinic keratosis. In vivo results also confirmed maximum AUC, t1/2, and skin-related drug retention of 5FU-Alg-Np-HG. (4) Conclusions: Chitosan-gelatin-based hydrogels containing 5FU-Alg-Np possess exceptional properties, and can be used for the sustained delivery of 5FU for the treatment of local skin cancers.(1) Background: Chitosan-gelatin-based thermosensitive hydrogel containing 5FU-alginate nanoparticles was formulated for the effective and sustained delivery of 5FU to the skin. (2) Methods: Alginate, a polysaccharide was used for the formulation of nanoparticles using a spray drying technique. Size, zeta potential, and surface morphology were investigated using a zetasizer and scanning electron microscope. The hydrogel was fabricated using chitosan and gelatin. Several important analyses were used to characterize these prepared topical hydrogels. The pH, visual transparency, rheological behavior, and swelling index of the prepared hydrogels were evaluated. The in vitro release studies were performed at different pH (5.5 and 7.4) and temperature (32 and 37 °C) conditions using a Franz diffusion cell. Ex vivo permeation and in vivo studies were performed using Sprague Dawley rats. (3) Results: Results show that spherical nanoparticles were produced at sizes of 202–254 nm and with zeta potentials of −43 to −38 mV. The prepared nanoparticles were successfully incorporated into chitosan-gelatin-based hydrogels using a glycerol 2-phosphate disodium salt hydrates crosslinker. Drug polymers and excipients compatibility and formulation of hydrogels was confirmed by ATR-FTIR results. The pH of the prepared hydrogels was in accordance with the skin pH. The viscosity of prepared hydrogel increased with temperature increase and phase transition (sol-gel transition) occurred at 34 °C. The release of drug was sustained in case of nanoparticles incorporated hydrogels (5FU-Alg-Np-HG) as compared to nanoparticles (5FU-Alg-Np) and simple hydrogels (5FU-HG) (ANOVA;  Hydrogels is one of the most important biomaterials. Hydrogels possess a highly porous 3D arrangement of interconnected and crosslinked polymers containing a high portion of aqueous phases or biological fluids (higher than 20%), due to which, its network is not dissolved through chemical and physical interactions ,2,3,4. HRecently, stimuli-responsive hydrogels are gaining great attention due to their ability to control the release of drugs and bioactive molecules. These types of hydrogels respond to several stimuli to change their physical, chemical, and/or mechanical properties to favor drug release at the target site . The devw/v and temperature 30–34 °C. Gelatin can form a high-molecular-weight network in the presence of a crosslinker that is capable of swelling [Nanoparticles (NPs) made from biodegradable natural polysaccharides are of great interest for the delivery of peptide, protein, and other drugs that require controllable release behavior . The natswelling .The chemotherapeutic agent,5-fluorouracil (5FU), is most frequently used in the treatment of cancer. However, its short biological half-life (5–10 min) is the main problem that limits its application. This is due to its fast metabolism by liver enzyme dihydropyrimidine dehydrogenase . AdditioTo improve the clinical efficiency of 5-fluorouracil, 5FU-alginatenanoparticles were prepared and incorporated into chitosan-gelatin hydrogels. Previously, different formulation techniques were used for local skin delivery, such as nanoemulsions, microemulsions, solid lipid nanoparticles, nanostructured lipid carriers, liposomes, and nanoparticles ,30. The t-test; p < 0.05), confirming the encapsulation of 5FU into the matrix of crosslinks formed by alginate chains in nanoparticles.The Alg-5FU nanoparticles were prepared using a spray drying technique;5FU can be encapsulated in the matrix of the polysaccharides via bonds with functional groups in the alginate chain . ParticlThe 5FU solution and 5FU-Alg-Np-loaded chitosan-gelatin-based hydrogels were prepared. Chitosan and gelatin solutions when mixed together, electrostatic interaction occurs, which results in interpenetrating self-assemble soft aqueous pores as a result of amide bonds formation . These s−1  [−1, and for alginate were at 3121, 2928, 1404, and 1050 cm−1. The characteristics peaks of gelatin were observed at 3292 cm−1 (N–H stretching), 2947 cm−1 (C–H stretching), and 1637 cm−1 (C=O stretching). The FTIR spectrum of nanoparticles have the characteristic absorption peaks at 3339, 2924, 1450, and 1060 cm−1, showing the absorption bands shift to new positions as compared 5FU. This indicates the successful encapsulation of 5FU into alginate nanoparticles. The absorption peaks of nanoparticles and 5FU were weaken in FTIR spectrum of hydrogels  . The chaydrogels . This coThe pH is an important parameter for topical formulations. The pH of all the prepared hydrogels was checked using digital pH meter . The pH G′) of the prepared hydrogel increase gradually after 30 °C, which confirms the thermosensitive behavior of the prepared hydrogels. On the other hand, the viscous modulus (G″) remained constant [G′ indicates mechanical reinforcement of the gel under body temperature (37 °C) conditions, showing a stronger hydrogel network [Rheological analysis can provide information regarding the ability of gel formulations to spread on the skin. The rheological properties of the gel can also affect the drug release kinetics and therefore, the study of these properties is essential. The viscosity of the prepared hydrogel remained higher between 15–36 °C, and then started to decline until 50 °C, showing the viscoelastic behavior of the hydrogels. Viscosity of nanoparticles incorporated hydrogels was higher than the simple hydrogels. This was due to the presence of polymer alginate in 5FU-Alg-Np-HG. The hydrogel becomes more viscous at body temperature as compared to the room temperature. Similar results were also previously reported by . Chitosaconstant . Higher  network .The surface morphology of the prepared hydrogel (5FU-Alb-Np-HG) was investigated using scanning electron microscopy. It was observed that the prepared hydrogels exhibited a dense and porous network structure as shown by red arrows in SEM image . The porSwelling is described as a phenomenon in which water and/or biological fluids are retained in a polymer network. Swelling ability of the prepared hydrogels was evaluated by monitoring the mass changes during incubation in the dissolution medium. The swelling studies of chitosan-gelatin-based hydrogels containing 5FU-Alg nanoparticles at temperatures 32 and 37 °C and pH at 5.5 and 7.4 were performed. The capability of a hydrogel to swell depends on its chemical structure and the characteristics of its medium. It was found that the swelling of hydrogel increases with increase in temperature from 32 to 37 °C . This mip < 0.05). In case of nanoparticles, it was found that rapid release of 5FU occurs during the first 120 min, followed by slower/sustained release. A much slower rate of release was observed in case of nanoparticles incorporated hydrogels with 45% release in 24 h. The initial burst release from the hydrogel might be due to weak absorption of the drug/nanoparticles in the hydrogel network. Furthermore, it was found that the release of 5FU was more sustained from the hydrogels at 37 °C as compared to 32 °C . This was due to higher swelling of hydrogels at higher temperature (37 °C). The release of 5FU from solution and nanoparticles did not change significantly when the temperature increased from 32 to 37 °C.The release of drugs from polymeric materials can be controlled by a number of physical processes, such as hydration, swelling, gel formation, drug diffusion from the matrix, and the erosion of the matrix . The relThus, the results indicate that the 5FU drug in the nanoparticles and hydrogels is highly protected from the physiological environment, and the release behavior from the nanoparticles and hydrogels is modulated by changes in environmental pH and temperature. It was noted that the drug release behavior from the chitosan-based hydrogels is similar to a previous study conducted by . A Korsmp < 0.05)  a. This wp < 0.05)  b. This wmax and Cmax values were observed for 5FU sol, this was due to inability of solution to penetrate the skin. The Cmax was slightly higher for 5FU-Alg-Np as compared to hydrogels. This was attributed to the smaller size, higher drug release and ex vivo permeation of nanoparticles. The half-life and AUC were found higher in case of 5FU-Alg-Np-HG as compared to other formulations. The longer half-life shows prolonged delivery of 5FU from the hydrogel in comparison to solution and nanoparticles. The t1/2 of the drug is an important parameter and it controls the degree of drug accumulation, concentration fluctuations and the time taken to reach equilibrium [p < 0.05). Higher skin-related drug retention is helpful in treating local skin cancers.The mean plasma drug concentration vs. time was plotted and is shown in ilibrium . FindingIn this study, 5FU alginate nanoparticles were prepared using spray drying technique. The prepared nanoparticles were 202–254 nm in size, had a zeta potential of −43 to −38 mV, drug content of 78%, and exhibited aspherical shape. The nanoparticles were incorporated into chitosan-gelatin hydrogels to prevent the premature release of 5FU at the surface of skin. The release of 5FU from nanoparticles was higher as compared to the hydrogels. This was attributed to the additional processes of diffusion of 5FU from the gel base. Furthermore, 5FU was released from the hydrogels using diffusion mechanism followed by swelling and erosion. The skin-related drug retention was significantly higher in case of hydrogels as compared to nanoparticles. The reason behind this is the higher swelling of hydrogels in lower skin layers, which helps in the prolonged retention of 5FU in the skin. Similar results for hydrogels were also observed for in vivo studies. Chitosan-gelatin hydrogels containing 5FU-alginate nanoparticles is an ideal delivery system for the treatment of localized skin cancer.In future, the developed hydrogel system can be evaluated for its efficacy against skin cancers, such as melanoma, using nude mice.The drug of interest, 5-flourouracil, wasobtained from Sigma-Aldrich (lot# A263299) . Alginate  was used for nanoparticle preparation. Chitosan  and gelatin  were used in hydrogel preparation. Acetic acid and methanol were obtained from Merck . All the chemicals used were of analytical grade.w/w) under continuous stirring at 1000 rpm. In addition, 0.3% w/w tween 80 was also added to the alginate solution as a surfactant. The 5FU (0.5% w/w) was taken and dissolved in distilled water, and the 5FU solution was then transferred to the alginate solution under constant stirring at 1000 rpm for 2 h. The drug polymer solution was then subjected to the spray dryer for nanoparticle preparation. The process parameters used were same as previously reported by our laboratory [The 5FU nanoparticles were prepared using a nano spray dryer . Alginate was used as a model polymer and dissolved in distilled water (1% boratory . The inlw/v) and determined on zetasizer in triplicate; the results were averaged.A DLS (dynamic light scattering) zetasizer  was used to measure the size and zetapotential of the prepared 5FU-Alg nanoparticles. The nanoparticles were taken and suspended/dispersed in 2 mL of ethanol (0.2% SEM (scanning electron microscope) (Zeiss supra 40 VP scanning electron microscope) was used to investigate the surface morphology of the prepared 5FU-Alg nanoparticles and hydrogels. The prepared nanoparticles were taken and mounted on aluminum stubs with the help of double-sided conducting carbon tapes. The stubs were then coated with a 50/50 mixture of Pt/Pdin order to minimize the surface charging. Finally, the samples were scanned at an accelerating voltage of 10 kV, and photographs with different magnifications were taken.A subsample of 15 mg of 5FU-Alg-Np was taken and dissolved in phosphate buffer pH 7.4 (30 mL) under continuous stirring at 1000 rpm for time period of 2 h. The sample was then taken, filtered using a syringe filter (0.45 µm), and then analyzed on a UV-Visible spectrophotometer  at wavelength 265 nm . The perv/v acetic acid solution. Glycerol 2-phosphate disodium salt hydrate (44.4% w/v) (GP)  solution was then added dropwise to the chitosan-gelatin solution under continuous stirring in an ice-water bath. The pH value of chitosan-gelatin solution was adjusted to 7.4 by adding GP solution. The chitosan-gelatin-GP solution was stored at 4 °C and utilized as a controlled release system for both 5FU and 5FUnanoparticles. Subsequently, 5FU and 5FU-NPs were added to the chitosan-gelatin-GP solution under stirring and cooled in an ice-water bath for 30 min. The hydrogel was placed in an ultrasonic bath for further 30 min to eradicate entrapped air bubbles. The developed hydrogels were stored at 4 °C until further use [Chitosan-gelatin hydrogels were prepared using previously developed method reported by Cheng et al., 2018 , with slther use .−1 (32 scans per minute with a resolution of 4 cm−1) in transmission mode. Three spectra of each sample were taken and the results were averaged.The ATR-FTIR analysis of 5FU, alginate, chitosan, 5FU-Alg nanoparticles and hydrogel were performed using PerkinElmer ATR-FTIR spectrometer . The sample was placed on zinc selenide, and the spectra was recorded in range of 600–4000 cmA digital pH meter  was used to measure the pH of the chitosan-gelatin hydrogel at a temperature of 25 °C. All measurements were performed in a triplicate and results averaged.Transparency of the chitosan-gelatin hydrogel was determined at different temperatures (25–37 °C). A transparent solution indicates that all the added ingredients are homogeneously mixed .The vial inversion method was used to measure the gelation time of the chitosan-gelatin hydrogel. The hydrogel components were mixed in a glass sample vial and incubated at 37 °C using water bath immediately after mixing and inverted every minute. The gelation time refers to the time required for “no flow” to be observed for hydrogel formulation .G′) and viscous modulus (G″) were analyzed at a gap of 1 mm and a fixed frequency of 1.0 Hz. Gelation temperature of the samples was analyzed in a range from 25 °C to 40 °C at a rate of 0.5 °C per minute. The sol-gel transition time of the samples was analyzed at 34 °C.The rheological properties of the prepared hydrogels (chitosan-gelatin-based hydrogel containing 5FU and 5FU-Alg-Np) were analyzed using a rheometer  (with a 60-mm diameter plate geometry with a gap of 1 mm). The sample of prepared hydrogel was added to the plate of rheometer at a temperature of 25 °C. The elastic modulus  at −50 °C for 24 h. The freeze-dried hydrogels were immersed in phosphate buffer saline (pH 7.4) at 37°C with shaking at 100 rpm. At predetermined time intervals, the hydrogels were removed from the PBS, the liquid was removed from the surface of hydrogel with tissue paper, and then the hydrogel was weighed .The swelling index was calculated using Equation (2);In vitro release studies were performed across synthetic membrane  using Franz diffusion cell. The donor compartment of the Franz diffusion cell was filled with drug polymer solution-nanoparticle-hydrogel and receptor compartment with release medium (phosphate buffer pH 5.5 and 7.4). The temperature of the medium was maintained at 32 and 37 °C. At specific time intervals , 2 mL of sample was collected and analyzed on UV spectroscopy at 265 nm . The in The ex vivo permeation study of prepared nanoparticles and hydrogels was performed on Franz diffusion cell using the rats’ skin as a membrane. The experiment was performed using the following working conditions: (i) receptor medium, phosphate buffer saline at pH 7.4; (ii) temperature, 37 °C ± 2 °C, (iii) rotation, 100 rpm. At predetermined time intervals, 2 mL of receiving solution was taken and replaced with fresh medium to maintain the sink condition. UV spectroscopy at 265 nm was used to determine the concentration of drug in the receiver medium.After the permeation experiment, skin was removed from the Franz diffusion cell and surface was cleaned with phosphate buffer saline. The skin (exposed area to permeation experiment) was cut into small pieces with scissor, and transfer to beaker containing 20 mL of PBS pH 7.4. Subsequently, 5 mL of methanol was added for homogenization of skin and to extract the 5FU from the tissues. The solution was centrifuged for 5 min at 5000 rpm. The supernatant was taken and drug was quantified by means of a UV spectrophotometer at 265 nm .Sprague Dawley rats were used for in vivo studies. The animal procedures were approved by the Ethical Review Board, Gomal University Pakistan. For in-vivo tests of 5FU-Alg-Np and Hydrogels,15 male Sprague Dawley rats, weighing 200–250 g, were used. The rats were divided into three groups , each group contains five rats (n = 5). The rats were anesthetized using ketamine/xylazine mixture, and then the dorsal region was shaved with sharp blades. After the application of formulations, 0.5 mL of blood sample was collected at specific intervals  from the retro-orbital vein. The plasma drug concentrations were quantified using high-performance liquid chromatography (HPLC) and our previously developed method . Pharmact-test and ANOVA, followed by post-hoc tests using Tukey’s honestly significant difference test, when required. The differences are considered significant at p < 0.05.All the experiments were performed in triplicate, and the results are presented as the mean values ± standard error (SE). The statistical analysis was performed using Student’s


Although the symptomatology has been assessed in multiple studies among persons recovering from coronavirus disease 2019 (COVID-19), less is known regarding long-term general health and disability. We aimed to assess long-term self-reported disability in public employees after predominantly mild COVID-19 in comparison with individuals who had negative COVID-19 polymerase chain reaction (PCR) test results.n = 56,221) and were invited to complete an online survey that included the World Health Organization Disability Assessment Schedule. Questionnaires were sent out between January 26 and March 5, 2021. A total of 14,222 (25.3%) employees responded, of which 10,194 (18%) were included . Of these, 7185 (70.5%) participants had a negative PCR test result (controls). A total of 1425 (14%) had a positive PCR result and were categorized in the sub-acute phase (4–12 weeks post COVID-19), and 1584 (15.5%) had a positive PCR test and were categorized in the post COVID-19 phase (> 12 weeks).Public employees within Region Västra Götaland were offered tests to identify SARS-CoV-2 infection (p < 0.001) as well as in a comparison between participants in the post COVID-19 phase and controls . Compared with controls, severe disability was more common in the post COVID-19 phase among both women , and men (9.8% vs. 6.8%).Fifty-two percent of controls rated disability of varying degrees, versus 73% and 64% of participants in the sub-acute and post COVID-19 phase, respectively. Being “emotionally affected” was the most frequently reported disability in the sub-acute phase, the post COVID-19 phase, as well as in controls. The proportion of participants reporting difficulties for 20–30 days was higher in the sub-acute phase than in the post COVID-19 phase (27.9% vs. 21.8%, Disability was present in all groups; however, reported disability was greater in the sub-acute phase than in the post COVID-19 phase. The higher levels of disability reported in the COVID-19 patient population may indicate a persisting need for rehabilitation and recovery. In general, women reported a greater degree of disability than men in the sub-acute and post COVID-19 phases.The online version contains supplementary material available at 10.1186/s12889-022-14820-3. Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By August 2022, nearly 600 million cases had been confirmed worldwide, with the equivalent number in Sweden approaching 2.6 million ; howeverThe level of exposure to SARS-CoV-2 in society is variable. Some occupations require personnel to come in close contact with other individuals (such as patients) whereas in other occupations, personnel have the option to work from home. Health care workers, particularly those working with patients who have COVID-19, have been identified as a group at high risk of exposure . FurtherThe International Classification of Functioning, Disability and Health (ICF) is a framework to describe health and health-related states . Althoughttps://www.1177.se, a Swedish online health care service driven by Inera AB on behalf of the regions of Sweden. It was not possible to submit the questionnaire without answering all questions. Two reminders were sent out, with the first sent 1 week after the first invitation. The study population has been described previously ), and comparisons between groups were performed with the Mann–Whitney U test. A two-tailed  scoring . The tot scoring . In partn = 41,999) showed that included participants were older  and comprised a larger proportion of women . Based on aggregated data regarding PCR test positivity in the eligible population, we estimate that the response rate was approximately 50% among participants with a positive PCR test and 20% among those with a negative PCR test.Drop-out analysis of non-participants ; and the mean age was 44.9 years (SD 12.1). Of these, 7185 (70.5%) participants had a negative PCR test, 1425 (14%) had a positive PCR test and were in the sub-acute phase, and 1584 (15.5%) had a positive PCR test and were in the post COVID-19 phase  as well as in comparison between participants in the post COVID-19 phase and controls . Furthermore, participants in the post COVID-19 phase reported having a reduced number of days in which they could participate in normal activities or work to a greater extent than controls ; however, this was reported to a lesser extent by the former group than by participants in the sub-acute phase   Fig. .Fig. 2DiRegarding the analysis according to sex, women reported a higher degree of disability than men in 5 items among controls, in 10 items among participants in the sub-acute phase, and in 7 items among participants in the post COVID-19 phase for WHODAS items 1–12   the number of patients reporting full recovery at 12 vs. 18 months was almost equivalent (39% and 38% respectively) [We report our study findings regarding the long-term effects on health and disability among public employees after predominantly mild COVID-19. We found that the majority of participants reported disability, but the prevalence was higher among participants in the sub-acute phase (73%) and post COVID-19 phase (64%) than among negative controls (52%). There was a lower prevalence of disability among participants in the post COVID-19 phase than in the sub-acute phase. This could be interpreted as an indication of a natural course of recovery. Conversely, in a sample of symptomatic patients from the general population (ctively) .The clinical case definition published by the WHO of post COVID-19 condition includes the phrase “a history of probable or confirmed COVID-19” . As the We found a greater level of disability in women than men. These results are in line with a previous report in a normative population showing that women report slightly greater disability than men, according to the WHODAS . AnalysiThe WHODAS covers all areas of the ICF . There iDisability is an umbrella term for impairment, activity limitation, and restricted participation ; therefoThe strengths of the present study include the relatively large sample size and the inclusion of a control group for comparison. Answers to questionnaire items were mandatory so there were no missing variables. The questionnaire contained one question regarding if the participants themselves thought that they had had COVID-19. This question helped us exclude participants from the control group who thought that they had had COVID-19, and therefore improving the selection of participants to the control group. We acknowledge the following limitations in the present study. First, the response rate was 25%, which is a limitation that entails a loss of power; however, approximately 50% of participants with a positive PCR test result were included in the study. Two reminders about the questionnaire were sent to increase participation. Antibody tests were offered to employees on a voluntary basis outside of the study. Therefore, we did not have data regarding this in all participants. Public employees within Region Västra Götaland were urged to get tested whenever they experienced symptoms (even mild ones); however, if the person nonetheless chose not to get tested and then reported “not having had COVID-19” they could have been misclassified into the group of persons with negative PCR results (controls). Asymptomatic persons could have been misclassified as controls and symptoms and disability in this group may therefore have been underestimated. Testing was not established in all work places until the fall of 2020 and access in the community was limited until summer 2020. Thus, some participants may have had unconfirmed COVID-19. Also, although we had access to all tests ordered by participants’ employers, access to tests ordered in routine care was limited primarily in one part of Region Västra Götaland (Skaraborg). Thus, some participants may have had confirmed COVID-19 without our knowledge. However, by also asking participants if they thought they had had COVID-19, we believe that we minimized the number of negative controls who had in fact had COVID-19. Presumably, the population with symptoms were more likely to complete the questionnaire. Furthermore, the study participants mainly comprised women of working age; therefore, our findings may not be generalizable to other populations. The time from PCR testing to completing the questionnaire varied; a longer follow-up increases the risk of recall bias regarding the degree of acute disease. Finally, the WHODAS is a generic instrument that has not been validated for use in patients with COVID-19; however, the WHODAS has been used previously to describe disability in patients with COVID-19 treated in the ICU .In the present study, disability was present in all groups. However, disability was greater in the sub-acute phase than in the post COVID-19 phase. The higher levels of disability reported in the COVID-19 patient population may indicate a persisting need for rehabilitation and recovery. In general, women reported a greater degree of disability than men in the sub-acute and post COVID-19 phase.Additional file 1: Supplementary Table 1. The number of participants reporting disability  on each WHODAS item (1–12). Supplementary Figure 1. Disability according to WHODAS, sex, test results and follow-up time after PCR testing. Supplementary Figure 2. Difference in number of days with difficulties according to sex, test results and follow-up time. Supplementary Figure 3. Total degree of disability according to WHODAS, sex, test results and follow-up time. Supplementary Figure 4. Total degree of disability with respect to occupation, test results, and follow-up time.


Follicular thyroid cancer (FTC) is a highly aggressive type of endocrine malignancy. It is necessary to investigate the mechanisms of tumorigenesis and therapeutic pathways in patients with FTC. Haem oxygenase-1 (HO-1) can regulate oxidative stress and the occurrence of tumors and diseases. In this study, we discovered that HO-1 was abnormally overexpressed in FTC compared with adjacent tissues. However, the HO-1 overexpression was demonstrated to decrease cell viability and to potentially activate the ferroptosis signalling pathway. Ferroptosis is a newly identified form of oxidative cell death and is currently being targeted as a new cancer treatment. Tumorigenesis is significantly inhibited by curcumin. The present study shows that curcumin inhibits the growth of FTC by increasing the HO-1 expression, further activating the ferroptosis pathway. This study demonstrates that the HO-1-ferroptosis signalling pathway might play an important role in FTC tumorigenesis, and that curcumin inhibits the growth of FTC cells by affecting this pathway. Thyroid cancer is the most common endocrine malignancy and the fifth most common cancer among women in the United States, and it ranks fifth among new cancer cases worldwide , 2. ThyrHaem oxygenase-1 (HO-1), a rate-limiting enzyme for haem degradation, can metabolize haem into biliverdin/bilirubin, carbon monoxide, and ferrous iron . MultiplResearch has demonstrated that curcumin has potential applications in pharmacology and cancer treatment . Curcumivia a mechanism that may involve the direct action of HO-1 and the ferroptosis signalling pathway.This study was designed to elucidate the molecular mechanism underlying the inhibitory effect of curcumin on FTC and to propose a new intervention for FTC. The results show that HO-1 is upregulated in FTC tumorigenesis. In addition, drug intervention experiments demonstrated that FTC tumorigenesis is inhibited by curcumin FTC samples and adjacent tissues were collected and immediately frozen in liquid nitrogen. All of the samples were collected with the written informed consent of all individual participants in this research, and all of the FTC samples were confirmed as FTC by pathologists through histopathological evaluation. The research was performed in accordance with the Declaration of Helsinki. The study was authorized by the Ethics Committee of the Second Hospital of Dalian Medical University.2 environments in incubators.Nthy-ori-3-1 , FTC-133, and FTC-238 (two types of FTC cells with different levels of invasiveness) cells were acquired from the Chinese Academy of Sciences' cell bank  along with Cell Line STR Authentication Reports. The Nthy-ori-3-1 cells were cultivated in RPMI 1640 with 10% foetal bovine serum (FBS). FTC-133 and FTC-238 cells were cultivated in DMEM with 10% FBS. All cells were cultivated in 37°C and 5% COThree pairs of samples, collected from the Second Hospital of Dalian Medical University, were sent to Novogene  for next-generation sequencing.Erastin (M2679), ferrostatin-1 , and deferoxamine  were all purchased from AbMole Bioscience Inc. . Curcumin  and autophagy inhibitor  were purchased from Sigma . Apoptosis inhibitor  was purchased from Beyotime .μm) were incubated with the primary antibody anti-HO-1  overnight at 4°C. The next day, the sections were incubated with goat anti-rabbit Envision System Plus-HRP for 30 min at room temperature. The sections were rinsed three times using phosphate-buffered saline (PBS) for 10 min each, and then diaminobenzidine (DAB) was used to incubate the sections for 2 min. The tumor sections were dehydrated and mounted after counterstaining with Mayer's haematoxylin. Finally, the sections were visualized by an Olympus microscope at a magnification of ×20. Two experienced pathologists examined the sections under a double-blind protocol. The results of immunohistochemistry were scored according to the percentage of positive cells and the intensity of cell staining. The scores for the percentage of positive cells were zero (no positive cells), one (≤25%), two (25–50%), three (51–75%), and four (≥75%), and those for the intensity of the cell staining were zero (negative for staining), one (weakly positive staining), two (moderately positive staining), or three (strongly positive staining). The final score for each specimen was the product of the two scores. Based on the arithmetic mean of these scores, the specimens scoring 8 or higher were deemed to have high expression.Human thyroid tumor specimens were acquired from the Department of Pathology. Tumor sections  lysis buffer. After resolving the proteins on 10% SDS-PAGE gels, the proteins were transferred onto PVDF membranes, which were incubated with the primary anti-HO-1 antibody  or anti-GPX4 antibody  at 4°C overnight. Next, the PVDF membranes were incubated with peroxidase-conjugated secondary antibody  for 2 h at room temperature. Western blotting results were calculated by using ImageJ software and normalized to the 3 cells per well). Each group was represented by 6 replicate wells. The plate was placed in an incubator at 37°C in a 5% CO2 environment. The cells were allowed to adhere. The medium was then replaced with medium including the specified concentrations of reagents, and the cells were incubated for 24 h. Each well was then supplemented with 10 μl Cell Counting Kit-8  solution, and then the 96-well plate was incubated at 37°C for 1 h. The absorbance at 450 nm was measured using a microplate reader .To evaluate cell viability, cells were digested by trypsin at the logarithmic growth phase and evenly added to a 96-well plate  was used to stain the cells after they were treated with methanol. Finally, the cells were photographed and counted in three random fields by using an Olympus microscope  at a magnification of ×20.Cells were transferred to the upper chamber of a transwell membrane filter  after resuspension in serum-free medium, and 600 Iron contents were detected by using an Intracellular Iron Colorimetric Assay Kit . Cells were collected with precooled PBS and then centrifuged at 12000 rpm for 10 min. The supernatant was collected for the following tests. GSH is an antioxidant, and MDA is an end product of lipid peroxidation. Both are markers of oxidative stress. A GSH assay kit  and an MDA assay kit  were purchased from Nanjing Jiancheng Bioengineering Institute. Cell samples were treated as described in “iron content detection” above. All operations were executed according to the manufacturer's instructions , 22. TheμM. PBS was then used to wash the cells three times. Finally, a fluorescence microscope  was used to acquire images at a magnification of ×40.Lipid ROS, considered one of the most important markers of ferroptosis, was measured by using Liperfluo . We seeded cells in 24-well plates, pretreated them with curcumin-containing medium for 24 h, and incubated them for 30 min in the dark with Liperfluo at a working concentration of 1 Small interfering RNAs (siRNAs) with sequences targeting HO-1 or GPX4 were constructed by GenePharma . ΔΔCT- method. Total RNA was extracted from cells using an RNA extraction kit . Evo M-MLV RT Premix  and SYBR Premix Ex TaqTM  were used to prepare the samples for RT-qPCR, which was conducted on a Rotor-Gene Q instrument . We calculated the relative expression levels of target genes based on the 2μl elution buffer two times. Finally, western blotting analyses were performed on the precipitated proteins.According to the manufacturer's instructions , the interaction between HO-1 and GPX4 protein was evaluated by Co-IP. Protein was extracted from cells by using IP lysis buffer supplemented with protease inhibitor. A BCA assay was used to measure the protein concentrations. The HO-1 antibody or IgG antibody was mixed with protein extracts and then incubated overnight at 4°C with incubation buffer. The following day, antibody-conjugated beads were used to immunoprecipitate HO-1 and its interacting proteins for 2 h at room temperature. Washing buffer was used to wash the protein-bead complexes five times, and then the protein was eluted with 40 t test. A chi-square test was used for immunohistochemistry analysis. p values less than 0.05 were considered statistically significant.GraphPad Prism software  was utilized for all statistical analyses. The results are presented as the means and standard deviations (SDs) of three replicates per group. For comparisons between different groups, experimental data were analyzed using one-way/two-way analysis of variance (ANOVA) or p < 0.05, q < 0.05, and the fold change . HO-1 mRNA levels were dramatically increased in FTC . Correspondingly, 33 pairs of thyroid cancer tissues and adjacent tissues were examined for the expression of HO-1 by using an immunohistochemistry assay   . The difvia western blotting assay . In addition, western blotting assay was used to examine the HO-1 expression in three types of cells: Nthy-ori-3-1, FTC-133 (poorly invasive), and FTC-238 (highly invasive) cells. The results showed that the HO-1 expression was much higher in FTC-238 cells than in FTC-133 cells or Nthy-ori-3-1 cells . Accordingly, we hypothesized that the aberrant expression of HO-1 may be involved in the progression of FTC.Similarly, a significant increase in the protein expression of HO-1 in cancer tissues compared to adjacent tissues was identified y N = 8, . In addi-1 cells . Accordi. In contrast, HO-1-overexpressing Nthy-ori-3-1 and FTC cells were more sensitive than the corresponding siHO-1-treated cells to erastin-induced death . These findings support the view that the high HO-1 expression may be related to the initiation of ferroptosis, especially in thyroid cells.Erastin, a classic ferroptosis inducer, can accelerate cell death through the accumulation of ROS . To inveFigures . In cont Figures . These f20) values for FTC-133 and FTC-238 cells were 9.35 μM and 10.56 μM, respectively. Correspondingly, the 50% growth-inhibitory concentration (IC50) values for FTC-133 and FTC-238 cells were 23.29 μM and 22.62 μM, respectively. However, the IC20 and IC50 values for Nthy-ori-3-1 cells were 32.25 μM and 92.36 μM, respectively. Of note, only 32, 64, and 128 μM curcumin significantly inhibited the viability of Nthy-ori-3-1 cells. These findings indicated that the FTC and Nthy-ori-3-1 cells differed in their sensitivity to curcumin treatment, and that low concentrations of curcumin inhibited the viability of the FTC cells without affecting that of the normal thyroid cells.Curcumin has anticancer effects in multiple types of solid tumors . To bettμM and 20 μM for follow-up tests. The results of the colony formation assays demonstrated that curcumin treatment dramatically reduced the number of colonies formed . The results indicated that the viability of FTC-133 and FTC-238 cells treated with 20 μM curcumin was obviously elevated in the presence of ferrostatin-1. Moreover, at a concentration of 10 μM curcumin, cell viability was significantly affected by several of the pharmacological inhibitors. We hypothesized that ferroptosis is the main cause of cell death after treatment with 20 μM curcumin. Therefore, 20 μM curcumin was used in the subsequent experiments.CCK-8 assays were used to detect cell viability to determine the main form of curcumin-induced cell death. FTC cells were either pretreated with ferroptosis inhibitor Fer-1 or DFO), autophagy inhibitor (CQ or 3MA), and apoptosis inhibitor (Z-VAD-FMK) for 2 h and then treated with curcumin for 24 h or subjected to curcumin treatment alone Figures  (Supplem or DFO, μM curcumin for 24 h, and the levels of indicators associated with ferroptosis were determined. The results showed that curcumin decreased GPX4 protein expression and increased HO-1 protein expression (Figures Although the findings indicated that curcumin-induced FTC cell death may be closely associated with ferroptosis, whether curcumin directly induces ferroptosis in FTC cells remained unclear. To investigate this possibility, FTC cells were treated with 0, 10, or 20  Figures . In addi Figures ; MDA con Figures . However Figures . Further Figures . Thus, wGPX4 is an important suppressor of ferroptosis. The regulatory relationship of GPX4 and HO-1 remains unclear. Our experimental study showed that knockdown of HO-1 promoted the GPX4 expression in curcumin-treated Nthy-ori-3-1 and FTC cells. However, the GPX4 expression was obviously inhibited when HO-1 was overexpressed Figures . We hypoCo-IP experiments were performed in FTC cells to confirm that HO-1 can specifically interact with GPX4 and regulate its expression . To confvia activation of the ferroptosis signalling pathway. By catabolizing toxic intracellular haem, HO-1 exerts antioxidant, antiapoptotic, and cytoprotective effects. The catabolites of haem degradation can facilitate the angiogenesis and metastasis of cancer cells, as well as their evasion of immune surveillance, thereby influencing cellular functionality and promoting tumor progression [FTC is an extremely aggressive malignant cancer of the thyroid and is more aggressive than PTC . In thisgression ; these pgression –30. ExpeμM curcumin on FTC cells, but it is still unclear whether curcumin suppresses FTC cells by triggering ferroptosis.Several early studies indicated that ferroptosis is not associated with autophagy. In contrast, recent research suggests that autophagy promotes ferroptosis by selectively degrading antiferroptosis regulators . A growiFerroptosis is a type of iron-dependent oxidative cell death  and is cvia HO-1-induced activation of the ferroptosis signalling pathway. Further research is needed to determine the specific mechanism underlying the effect of curcumin on FTC; however, for patients who cannot tolerate surgery or are difficult to diagnose, it may be possible to develop a therapeutic pathway towards the remission of FTC based on our findings.We conclude that curcumin inhibits the tumorigenesis of FTC


The development of the n-type FTE is still at a relatively early stage with limited applicable materials, insufficient conversion efficiency, and issues such as an undesirably high cost or toxic element consumption. In this work, as a prototype, a flexible n-type rare-earth free skutterudite (CoSb3)/poly-polystyrene sulfonate (PEDOT:PSS) binary thermoelectric film was fabricated based on ball-milled skutterudite via a facile top-down method, which is promising to be widely applicable to the hybridization of conventional bulk TE materials. The polymers bridge the separated thermoelectric particles and provide a conducting pathway for carriers, leading to an enhancement in electrical conductivity and a competitive Seebeck coefficient. The current work proposes a rational design towards FTE devices and provides a perspective for the exploration of conventional thermoelectric materials for wearable electronics.Alongiside the growing demand for wearable and implantable electronics, the development of flexible thermoelectric (FTE) materials holds great promise and has recently become a highly necessitated and efficient method for converting heat to electricity. Conductive polymers were widely used in previous research; however,  ZT = S2σTκ−1, where S represents the Seebeck coefficient, σ represents electrical conductivity, κ represents thermal conductivity, and T represents absolute temperature. To improve the figure of merit, the majority of recent research is focused on two aspects: the enhancement of the power factor (S2σ) [κ [Out of the primary energy sources such as feedstocks, oil, natural gas, etc., there has been an increasing demand for the development of renewable and sustainable sources of energy ,2,3. Heaor (S2σ) ,15,16,17(S2σ) [κ ,25,26,27p-type TE performance. Benzodifurandione-based polyphenylene vinylene (BDPPV) [n-type TE performance. However, the n-type conductive polymers have a lower TE performance and stability in air compared to p-type polymers due to their unstable dopants [p-type polymers are still the mainstream in current FTE development. Among the various polymers, poly polystyrene sulfonate (PEDOT:PSS) stands out in particular because of its high electrical conductivity of up to 4839 S/cm [In recent years, a variety of novel flexible electronic devices, ranging from wearable smart electronics to printable circuit boards, have steadily been developed in line with the concept of the Internet of things (IoT) society ,29. The  (BDPPV)  and poly (BDPPV)  as condu dopants . Therefo839 S/cm , and the839 S/cm ,43. Desp839 S/cm ,44, whicn-type FTE film [n-type inorganic TE materials on flexible substrates such as nylon and/or PI substrates by suction filtration [xZn1-xFeS2) and PEDOT:PSS on the polytetrafluoroethylene (PTFE) membrane have been reported and exhibit outstanding flexibility and performance [Various methods have been conducted to build a hybrid composed of inorganic TE materials and polymers. For example, polyethyleneimine (PEI) has been used as a dopant in carbon nanotube yarn (CNTY) by donating electrons to fabricate an FTE film . Furtherltration  or magneltration  could alformance . This ve3, is another family of promising TE candidates which shows high TE performance in the medium to high-temperature range due to its high electrical conductivity [3 with Te to form a ternary solid solution alloy, Te provides electrons as a donor, and the electrical conductivity increases rapidly due to the increase in the carrier concentration [3-xTex has been mainly studied as a bulk TE material, the study of CoSb3-xTex as an FTE film will provide new insights and is expected to broaden the selection of inorganic materials used in the development of FTE materials in the future.Skutterudite, with a general formula of CoSbuctivity . By dopintration . Since Cn-type flexible CoSb3-xTex/PEDOT:PSS TE film was produced using a facile method. CoSb3-xTex ingots were prepared using a top-down method by breaking down the size of the bulk counterpart via ball-milling that does not require chemical treatment, and the powder was mechanically mixed with PEDOT:PSS, followed by suction filtering onto a membrane. The TE properties  with different compositions and Te doping of the hybrid films were evaluated and discussed, which demonstrated the potential for CoSb3-xTex to be developed into an FTE component.In this study, an Analytical grade Co  and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Analytical grade Sb  and Te  were purchased from Kojundo Chemical Laboratory corporation. PEDOT:PSS  was purchased from Heraeus Clevios. Mixed cellulose ester (MCE) membrane filter (with an average pore diameter of ~0.22 µm) was purchased from AS ONE corporation. All reagents were used directly without purification.3-xTex  was synthesized by a solid-state reaction. Co pieces, Sb grains, and Te grains were well-mixed in a stoichiometric ratio and added into quartz tubes that were subsequently sealed under vacuum conditions (1 × 10−3 mbar). The tubes were put into a muffle furnace, which was heated to 1423 K with a ramping rate of 0.8 K/min and kept for 24 h. The samples were quenched in water and annealed at 873 K for 96 h. The as-prepared ingot samples were ground with a mortar to obtain the powder, and the powder was ball-milled for 1 h by the 8000D Mixer/Mill.CoSb3-xTex skutterudite/PEDOT:PSS film is shown in 3-xTex powders were added to ethanol and sonicated for 30 min. A CoSb3-xTex dispersion with various skutterudite contents  was added to the PEDOT:PSS/DMSO solution and sonicated for 30 min. The mixture was vacuum-filtrated with an MCE membrane filter. After the film was dried in a vacuum oven at 60 °C for 10 h, the film was cold-pressed for 5 min at 20MPa. All of the TE properties were measured using MCE membrane-based hybrid films.The fabrication process for the CoSb2 atmosphere. The calibration was performed with an empty aluminum crucible. The evaluation of the thermoelectric properties including the electrical conductivity (σ), the Seebeck coefficient (S), and the power factor (PF) was carried out on a ZEM-3 . The measurement was performed in a standard four-probe configuration under a partial helium atmosphere (0.1 MPa) at RT.The morphology and elemental mapping was obtained from a field-emission scanning electron microscope  with an energy-dispersive X-ray spectrometer . In the SEM observation, a small piece of as-prepared film was pasted onto the SEM stage with carbon conductive tape. The accelerating voltage was 10.0 kV. The phase structural information was acquired from the X-ray diffractometer  with Cu Kα radiation (λ = 0.154 nm). The measurement was conducted under a voltage of 40 kV and a current of 40 mA  in the Theta-2 Theta geometry. X-ray powder diffraction patterns were refined via Rietveld analysis using the FullProf and WinPLOTR software packages ,52. The 2.95Te0.05 powder with the corresponding EDX spectrum presenting the elemental Co, Sb, and Te distribution in 3-xTex powders with different amounts of Te dopants are shown in 3 composition, which confirms the purity of the CoSb2.95Te0.05 prepared in this study. All the patterns can be indexed to the skutterudite structure (JCPDS no.01-083-0055) with a space group of Im-3 (No.204). The Rietveld refinement results are summarized in 3 structure purity is exempt from structural defects. Moreover, the lattice size varied linearly with the Te content attesting to the successful substitution of the Sb by the Te within the structure. 2.95Te0.05/PEDOT:PSS hybrid film. According to the digital photo image of the 98 wt% CoSb2.95Te0.05/PEDOT:PSS hybrid film shown in 2.95Te0.05 powder. As displayed in 2.95Te0.05 powder is very high. The morphology of the hybrid CoSb2.95Te0.05/PEDOT:PSS films with different fractions of PEDOT:PSS can be found in the SEM images in 2.95Te0.05 particles become more obvious with the increase in the weight percentage of the inorganic components. From the top view image of the 98 wt% CoSb2.95Te0.05/PEDOT:PSS hybrid film shown in 2.95Te0.05 particles can be observed with sizes ranging from submicron to several microns spread on the surface of the hybrid film. In the EDX mapping  and of skutterudite hybridized with different mass ratios of X CoSb3-xTex/1-X PEDOT:PSS  have been developed, and the influence on the FTE properties was investigated as shown in 3-xTex doping ratio and obtained a largest negative value of −161.7 μV/K at 98 wt%, considering an uncertainty of 6% from the measurement [3-xTex bulk sample, where the Seebeck coefficient increases with a decreasing doping rate due to the charge carrier tuning  of Te, CoSb2.95Te0.05, at 98 wt% shows the largest power factor of 6.47 µW/m K2 at room temperature.To optimize the CoSbsurement . This trr tuning . In othen-type flexible thermoelectric materials reported so far. In this study, CoSb2.95Te0.05/PEDOT:PSS hybrid film records the largest negative Seebeck coefficient value of −161.7 µV/K at ambient temperature. However, the electrical conductivity and the power factor are much lower compared to the previous work. We will improve our efforts in the future by downsizing the skutterudites and uniformizing the grain size. In addition, we measured the temperature-dependent TE properties of the 98wt% CoSb2.95Te0.05 hybrid film as shown in 3, which agrees with a large carrier concentration and supports the idea that the inorganic component led the electrical transport properties rather than the organic component.2.95Te0.05/PEDOT:PSS hybrid film, which has the highest thermoelectric performance, was subjected to a bending test to measure its flexibility as shown in The 98 wt% CoSb3-xTex/PEDOT:PSS hybrid film was produced using a simple method. First, we succeeded in our preliminary step to obtain a submicron to several micron particle size suitable for hybridization with the organic counterpart. Then, by suction-filtering the mixed solution with the PEDOT:PSS aqueous dispersion to an MCE membrane, we achieved the production of a uniform and flexible n-type FTE film. As for the TE performance, the Seebeck coefficient of the hybrid film increased as the amount of CoSb3-xTex in the hybrid film increased and the doping rate of CoSb3-xTex decreased. As a result, the CoSb2.95Te0.05/PEDOT:PSS hybrid film obtained the largest negative Seebeck coefficient of −161.7 µV/K at 98 wt% at room temperature. As it is more challenging to obtain a large negative Seebeck coefficient in the hybrid thin films than improving the electrical conductivity of these type of films, wherein few approaches have been attempted, our work established a useful method of improving the power factor by raising the Seebeck coefficient. In addition, the 98 wt% hybrid film retained the flexibility to maintain a certain degree of electrical conductivity even after being bent 1000 times. It was proved that it is relatively easy to fabricate a flexible film with a very small amount of PEDOT:PSS and obtain moderate properties by finely tuning the composition of the TE films. The same method in this study can be applied to other inorganic materials. Therefore, it is expected that the selection of inorganic TE materials used for the development of n-type flexible thermoelectric materials will be expanded in the future.In this study, a CoSb